Summary of work: The nuclear matrix is a central structure in the cellular hierarchy. A number of processes have been shown to be located here, including transcription, replication and topological binding sites. There has also been documentation that DNA repair processes are preferentially located at this site. We have demonstrated that the early DNA repair processes of nucleotide excision and transcription coupled DNA repair take place here at the nuclear matrix. In response to UV irradiation of mammalian cells, the protein proliferating cell nuclear antigen (PCNA) forms an insoluble complex with nuclear substructures. This complex can be detected by immunofluorescence and western blot analysis within 30 min after UV irradiation. We have studied the role of nucleotide excision repair (NER) and its subcomponent, transcription coupled repair (TCR), in PCNA complex formation. PCNA complex formation was studied in genetically related hamster cell lines that differ only in their capacity to perform NER. The hamster cell lines UV5 and UV24, which are homologs of the human DNA repair mutants xeroderma pigmentosum (XP) groups D and B, are completely deficient in NER. The hamster cell line UV61 is deficient in TCR of UV induced pyrimidine dimers, and is homologous to the human DNA repair mutant Cockayne syndrome (CS) complementation group B (CS-B). In the NER deficient cells, UV5 and UV24 cells, the PCNA complex was not detectable within 30 min after UV. When the UV5 cells were transfected with the human XPD gene, the PCNA complex formation was restored to normal. In the TCR defective UV61cells, the rate of PCNA complex formation was intermediate between normal and NER deficient cells. This defect in UV61 cells was complemented by transfection of the human CSB gene. We conclude that efficient PCNA complex formation induced by UV irradiation is dependent on both the genome overall repair of 6-4 photoproducts and the TCR of pyrimidine dimers in hamster cells. The complex formation occurs only in non-replicating cells and is not affected by pretreatment with aphidicolin. We have investigated the DNA repair of photo lesions in the ribosomal DNA in relation to the RNA polymerase I transcription in the nucleoli using a combination of immunological and biochemical approaches. Labeling of DNA repair sites with BudR in UV treated cells at different post incubation times showed a lack of DNA repair in the nucleolar regions comprising rDNA genes. Immunofluorescent labeling of UV induced repair and of transcription sites in interphase nuclei of hamster cells indicates that the repair of photo lesions in the rDNA genes is not coupled to transcription. The nucleoli are completely deficient in repair despite the presence of abundant RNA pol I transcription foci. Gene specific repair assays showed that both cyclobutane pyrimi-dine dimers and 6-4 photoproducts are removed much less efficiently from the rDNA than from an endogenous essential gene. This observation is further substantiated by the analysis of repair kinetics of UV induced photolesions in the isolated BudR containing repair patches isolated immunologically using antibody to BudR. Immunological staining indicates that various NER proteins like TFIIH (Subunits p62 and p89), p53, Gadd 45 and proliferating nuclear antigen are all enriched in the nuclei but distinctly absent in nucleoli. The lack of enrichment of NER factors in the nucleolus may be responsible for the inefficient repair of photoproducts in the rDNA.